Method for the identification of the clonal source of a restriction fragment

1. Method for the generation of a physical map of at least part of a genome comprising the steps of:

(a) providing a sample DNA;

(b) generating an artificial chromosome clone bank wherein each artificial chromosome clone contains part of the sample DNA;

(c) combining the artificial chromosome clones in a plurality of pools, wherein each clone is present in more than one pool of the plurality of pools, to create a library;

(d) digesting the DNA contained in the plurality of pools with one or more restriction endonucleases to provide for a set of restriction fragments for each pool;

(e) ligating adaptors to one or both sides of the restriction fragments, wherein at least one adaptor contains a pool-specific identifier or a degenerate identifier section, respectively, to provide adaptor-ligated restriction fragments;

(f) combining the adaptor-ligated restriction fragments of step (e);

(g) amplifying the adaptor-ligated fragments with at least one primer to provide amplicons;

(h) determining the sequence of at least the pool-specific identifier and part of the restriction fragment of the amplicons;

(i) assigning the restriction fragment sequences determined in the amplicons to the corresponding clones using the pool-specific identifiers;

(j) building a contig based on sequence matching of the restriction fragment derived sections; and

(k) ordering the restriction fragments of step (i) to thereby build a clone-contig and generating a physical map.

2. Method according to claim 1 , wherein the primer contains a pool-specific section corresponding to the pool-specific identifier section in the adaptor or contains a pool-specific identifier at the position of the degenerate identifier section, respectively, to provide amplified adaptor-ligated restriction fragments or amplicons.

3. Method according to claim 2 , wherein the amplified adaptor-ligated restriction fragments or amplicons are combined in a set of combined amplicons.

4. Method according to claim 1 , wherein the restriction fragments are assigned to the corresponding clone by clustering adapter-ligated restriction fragments or amplicons that contain identical sequences in the restriction fragments but carry different pool-specific identifiers.

5. Method according to claim 1 , wherein the sequencing is carried out by means of high-throughput sequencing.

6. Method according to claim 5 , wherein the high-throughput sequencing is performed on a solid support.

7. Method according to claim 5 , wherein the high-throughput sequencing is based on Sequencing-by-Synthesis.

8. Method according to claim 5 , wherein the high-throughput sequencing comprises the steps of:

annealing the adapter-ligated restriction fragments or amplicons to beads, each bead annealing with a single adapter-ligated restriction fragment or amplicon;

the beads in water-in-oil micro reactors, each water-in-oil icro reactor comprising a single bead;

performing emulsion PCR to amplify adapter-ligated restriction fragments or amplicons on the surface of heads;

optionally, selecting/enriching beads containing amplified amplicons;

loading the heads in wells, each well comprising a single head; and

generating a pyrophosphate signal.

9. Method according to claim 5 , wherein the high-throughput sequencing comprises the steps of:

annealing the adapter-ligated restriction fragments or amplicons to a surface containing first and second primers or first and second primer binding sequences respectively,

performing bridge amplification to provide clusters of amplified adapter-ligated restriction fragments or amplified amplicons,

determining the nucleotide sequence of the amplified adapter-ligated restriction fragments or amplified amplicons using labelled reversible terminator nucleotides.

10. Method according to claim 1 , wherein the identifier is from 4-16 bp.

11. Method according to claim 10 , wherein the identifier is from 4-10 bp.

12. Method according to claim 11 , wherein the identifier is from 4-8 bp.

13. Method according to claim 12 , wherein the identifier is from 4-6 bp.

14. Method according to claim 10 , wherein the identifier does not contain 2 or more identical consecutive bases.

15. Method according to claim 10 , wherein for two or more clones, the corresponding identifiers contain at least two different nucleotides.

16. Method according to claim 1 , wherein at least one primer carries 1-10 selective nucleotides at its 3′ and to provide for a subset of amplicons.

17. Method according to claim 16 , wherein the at least one primer carriers 1-4 selective nucleotides at it 3′ end.